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Natural Product Sciences
22(3) : 168-174 (2016)
http://dx.doi.org/10.20307/nps.2016.22.3.168
168
Modulation of Melanin Synthesis by Amaranthus spp. L Seed Extract
in Melan-a Cells
Jae Ok Seo1, Moon Ho Do1, Jae Hak Lee2,Taek Hwan Lee3, Hussain Mustatab Wahedi1,
Yong Un Park1, and Sun Yeou Kim1,4,5,*
1College of Pharmacy, Gachon University, Yeonsu-gu, Incheon, Republic of Korea2Korea Plant Resource Institute, Paju-si, Gyeonggi-do, Republic of Korea
3College of Pharmacy, Yonsei University, Yeonsu-gu, Incheon, Republic of Korea4Gachon Medical Research Institute, Gil Medical Center, Incheon, Republic of Korea
5Gachon Institute of Pharmaceutical Science, Gachon University, Yeonsu-gu, Incheon, Republic of Korea
Abstract − Anti-melanogenic effects of amaranth (AT), one of the key source of squalene, were investigated inmelanocytes. Amaranth seed powder was extracted with water and melan-a cells were treated with variousconcentrations of AT. By using HPLC, content of myo-inositol, one of potential active components, was measuredin the crude extract of AT.AT reduced the melanin content in melan-a melanocytes and down-regulatedmelanogenic enzyme activity such as tyrosinase, TRP-1 and TRP-2. By regulating melanogenic enzyme activity,AT may be a potential natural source for whitening agent. Myo-inositol was detected in AT by HPLC and may beone of the active compounds from AT involved in the regulation of anti-melanogenesis. In this study, wedemonstrated that AT has anti-melanogenesis properties. This new function of amaranth may be useful in thedevelopment of new skin-whitening products and its value as food. Keywords − Amaranth, Myo-inositol, Melan-a cells, Melanogenesis
Introduction
Melanosomes are biosynthesized in melanocytes in the
basal layer of the epidermis. They contain melanin that
protects the skin against harmful environments.1-3 Mela-
nosomes are transferred to keratinocytes through their
dendrites and accumulate in the form of granules in the
nucleus.4 Although melanin plays important roles in
melanogenesis, melanin overproduction can also cause
hyper-pigmentation, melasma, freckles, and skin cancers.
Therefore, the use of natural products that protect the skin
is a common issue in dermatologic clinics and cosmetic
research.5
Melanin is produced via several melanogenic enzymes,
including tyrosinase, tyrosinase-related protein 1 (TRP-1),
tyrosinase-related protein 2 (TRP-2), and microphthalmia-
associated transcription factor (MITF).6 MITF presents two
binding sites, M-box and E box, stimulates the expression
of melanogenic genes, and regulates the expression of the
alpha melanocyte-stimulating hormone (α-MSH) and the
cAMP pathway.7,8 When α-MSH is stimulated in melano-
somes, tyrosinase, TRP-1, and TRP-2 are activated.9,10
Especially, tyrosinaseis an important enzyme in the melanin
synthesis pathway because it triggers the melanogenic
process.11 It is responsible for the hydroxylation of L-
tyrosine to L-β-3,4-dihydroxyphenylalanin (DOPA) and
oxidation of DOPA to DOPA quinone.12,13 DOPA quinone
has the ability to transform melanin to pheomelanin or
eumelanin.14 TRP-2 serves as a dopachrometautomerase
and inverts dopachrome to 5, 6-dihydroxyindole-2-carboxylic
acid (DHICA). Subsequently, TRP-1 oxidizes DHICA to
indole-5,6-quinone-2-carboxylic acid, a precursor of eume-
lanin.8,14-16 Together with other melanogenic enzymes and
factors, tyrosinase is one of the main enzymes for the
development of skin whitening agents.9
Natural products cover traditional medicines and herbs.
They garnered attention in the cosmetic market because
they are safe and do not present side effects.17 Amaranthus
spp. L. (AT) has long been used as a dietary antioxidant
throughout South America and Asia, including Korea,
because it contains various amino acids and other
important micro nutrients.18-21 It is called the perfect food
*Author for correspondenceSun Yeou Kim, College of Pharmacy, Gachon University, 191 Ham-bakmoero, Yeonsu-gu, Incheon 406-799, Republic of Korea.Tel: +82-32-899-6411; E-mail: [email protected]
Vol. 22, No. 3, 2016 169
because it contains 60% starch, 8% fat with squalene, and
15% protein with a high concentration of lysine. Additio-
nally, it is a gluten-free food.22-24 Squalene is also widely
used as an antioxidant, skin moisturizer, treating skin
disorders like acne, psoriasis and atopic dermatitis.
Therefore, it is needed to find alternative natural resources
which explore the utility of squalene for skin cosmetics.
Squalene, a highly unsaturated hydrocarbon, found as an
important component of the liver oil of certain varieties of
deep sea fish. Cosmetic company wants to use plant
source to safe while actually deep sea products may not
be a good source for cosmetics because of significant
chemical contamination and toxicity such as mercury.
Amaranth which contains about 7% lipids seems to be the
key source of squalene.
Ultra violet radiation (UVR) stimulation induces the
production of reactive oxygen species (ROS) and causes
melanogenesis.25 Therefore, anti-oxidative natural products
may act as anti-melanogenic agents. The antioxidant
activities of extracts from amaranth leaves, flowers, and
seeds were previously reported.26,27 We are to focus the
research into the anti-melanogenic effects of amaranth for
skin cosmetic product, which is still in its early stages.
Therefore, our study was designed to investigate the anti-
melanogenic activity of AT in melanocytes.
Experimental
General experimental procedures – RPMI 1640 was
purchased from Gibco-BRL (Gaithersburg, MD, USA).
Fetal bovine serum (FBS) and penicillin-streptomycin
(PS) were purchased from Hyclone (Carlsbad, CA, USA).
Phenylmethylsulfonyl fluoride (PMSF), 12-O-tetradeca-
noylphorbol-13-acetate (TPA), 1-phenyl-2-thiourea (PTU),
kojic acid, triton X-100, aprotinin, mushroom tyrosinase,
3,4-dihydroxy-L-phenylalanin (L-DOPA), dimethyl sulfoxide
(DMSO), anti-α-Tubulin antibody were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). Anti-MITF
and anti-tyrosinase antibody were obtained from Cell
signaling (Danvers, MA, USA). Anti-TRP-1, anti-TRP-2
antibody, Horseradish peroxidase (HRP)-conjugated donkey
anti-goat IgG and goat anti-rabbit were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA, USA). Hor-
seradish peroxidase (HRP)-conjugated goat anti-mouse
IgG was purchased from Thermo scientific (Waltham,
MA, USA). Santa Cruz Biotechnology)
Amaranth seed extract (AT) – Each variant of amaranth
seed species was named as follows: Amaranth uscruentus
(AA), Amaranth uscruentus var (AB), Amaranth ushybridus
(AC), and the crossbreed between Amaranth ushybridus
and Amaranth ushypocondriacus (AD). Amaranth seeds
were air dried for 3 days at 40 oC to maintain their ger-
minative power. Powdered amaranth seeds were extracted
3 times in distilled water. After freeze drying, the extract
was fractionated with hexane and ethyl acetate using a
separating funnel. The steps were present in Fig. 1.
HPLC analysis – Analysis was carried out on a Waters
system (Waters Corp., Milford, MA, USA), consisting of
separation module (e2695). Evaporative light scattering
detector (2424) was used to develop a method for content
determination of myo-inositol in AD. Separation was
carried out using an Asahipak NH2P-50 4E (250 × 4.6
mm; particle size, 5 um; SHOWA DENKO K.K., Japan)
and Column temperature was maintained 30 oC. The
mobile phase composed water and acetonitrile (25:75, v/
v) and the flow rate was 1 ml/min. The temperature of
drift tube was 80 oC with nitrogen as developing solvent.
Cell culture – Mouse melan-a cells were provided by
Dr. Byeong Gon Lee at the Skin Research Institute, Amore
Pacific Co. (Yongin, Korea). RPMI 1640 supplemented
with 10% FBS, 1% PS, and 400 nM TPA was used to
maintain melan-a cells. All cells were incubated at 37 oC
in a humidified incubator with 5% CO2.
Measurement of the melanin content – Melan-a cells
(10,000/per well) were seeded in a 24-well plate and
incubated for 72 h after being treated with AT. After 72 h,
the melanin content was measured using the method
reported by Hosoi et al. with slight modifications.28 Briefly,
after removing the medium, the cells were washed twice
Fig. 1. Scheme of sample preparation. Simply present the stepsof extraction of AT. A: Amaranthus spp. L Total extract, B: Hexanesoluble fraction, C: Residue, D: EtoAc soluble fraction.
170 Natural Product Sciences
with phosphate-buffered saline (PBS). One milliliter of
sodium hydroxide solution (1 N) was then added to each
well to dissolve the melanin content. The melanin absor-
bance was measured at 405 nm using a microplate reader.
Measurement of cell viability – Cell viability was
determined by a 3-[4, 5-dimethylthiazol-2-yl]-2,5-dip-
henyltetrazolium bromide (MTT) assay. Melan-a cells
were seeded at 1 × 105 cells/well in a 24-well plate and
incubated for 72 h after being treated with AT for 24 h.
After 72 h, the medium was removed and 400 µL of
DMSO was added and the plates were placed on a shaker
for 10 min. The absorbance was then measured at 570 nm
using a microplate reader.
Tyrosinase activity assay in melan-a cells – Melan-a
cells were seeded at 10 × 105 cells in 100 mm dishes and
incubated for 72 h. Cells were detached by using trypsin-
EDTA and centrifuged. Tyrosinase buffer (80 mM phosphate
buffer + 1% Triton-X 100 + 100 µg/mL PMSF) was added
to the cell pellets for 1 h and centrifuged at 12,500 rpm
for 20 min at 4 oC. The supernatant was used to assess the
activity of tyrosinase. Cell lysate was measured to estimate
the protein content using bovine serum albumin (BSA) as
a standard; 150 µg of proteins were required for the reaction.
Tyrosinase activity was assessed by measuring the rate
of L-DOPA oxidation according to the method reported in
previous paper.16 To estimate the inhibitory effects of AT
on tyrosinase from melan-a cells, 0.1, 1, and 10 µg/mL of
AT and kojic acid (positive control) were dissolved in
methanol; 40 µL of each sample was then added to the
wells of a 96-well plate with 120 µL of L-DOPA and 150
µg of protein. The samples were mixed, incubated for 15
min, and the absorbance was measured at 490 nm using a
microplate reader.
Western blot analysis – 10 × 105 Melan-a cells were
seeded in 100 mm dishes and treated with 0.1, 1, and 10
µg/mL of A.C and A.D for 3 days. Cells were washed
with PBS and harvested using trypsin-EDTA. Detached
cells in 1 mL of PBS were centrifuged at 7500 rpm for 5
min and PBS was removed. Cell pellets were lysed using
lysis buffer (50 mM Tris-Cl, pH 8.0, 0.1% SDS, 150 mM
NaCl, 1% NP-40, 0.02% sodium azide, 0.5% sodium
deoxycholate, 100 µg/mL PMSF, and 1 µg/mL aprotinin)
for 1 h in cold conditions. After centrifugation at 12,500
rpm for 20 min at 4 oC, the supernatant was used for
western blot analysis. Protein contents were measured by
using BSA as a standard. Forty micrograms of protein
were separated by 12% SDS-PAGE and transferred to a
nitrocellulose membrane. The membrane was blocked for
1 h with 5% of skim milk in Tris-buffered saline-T
(TBST) and incubated overnight with α-tubulin (1:3000
dilution), MITF (1:500 dilution), tyrosinase (1:500), TRP-
1 (1:500 dilution), and TRP-2 (1:500 dilution) primary
antibodies at 4 oC. After removing the primary antibody,
membranes were washed 3 times with TBST and
incubated with the appropriate secondary antibody for 1
h. The bands were detected with a ChemidocXRS +
imaging system (Bio-Rad, Hercules, CA, USA).
Statistical Analysis – All experiments were repeated in
at least three assay for ELISA and western blot. All
values were presented as the mean + SD. The statistical
significance of difference between the data from the dose-
dependent assay was evaluated by student t-test, one-way
analysis of variance (ANOVA). A p-value < 0.05 was
considered statistically significant.
Result and Discussion
Amaranthus spp. L. may grow naturally or be cultivated.
The plant is known to contain any nutrients, minerals,
amino acids, and fibers. It shows anti-cancer properties,
prevents cardiovascular diseases, and lowers the risk of
diabetes.29 Furthermore, there are many reports of its
antioxidant activity.19, 30-32 However, until now, there is no
report on the whitening effect related to the antioxidant
activity of amaranth plants. Currently, the Amarantus
genus includes over 70 species, although the number of
species is questionable due to the hybridization concept
and technique. Our collaborator cultivated some amaranth
geneous. For the purpose of expanding the use of amaranth
cultivated in Korea, we first tried to develop amaranth
products for the cosmetic industry.
In order to examine the skin-whitening effect of amaranth,
we screened the effects of amaranth sub-species on the
melanin content in melan-a cells. In an attempt to
standardize amaranth samples, we measured myo-inositol
in each sample by HPLC analysis. The linearity of myo-
inositol was calculated by three concentrations. HPLC
data for myo-inositol and the extract of Amaranthus seeds
were shown in Fig. 2. The main component was myo-
inositol and its highest amount was found in AD
(49.036 ± 1.0325% ug/mg extract). Myo-inositol has been
reported to inhibit melanin synthesis by reducing the
activity of tyrosinase.33 As shown in Fig. 2, the level of
myo-inositol from sample is 5%. It means AT can be the
potential inhibitor of tyrosinase because it highly contains
myo-inositol. The main component of amaranth oil is
squalene. It is composed of polyunsaturated lipids which
is one of main component of skin surface. Therefore,
amaranth oil may show advantage for usage as a natural
base ingredient of cosmetics. Recently, it seems like the
Vol. 22, No. 3, 2016 171
cosmetic biotech are exploiting amaranth to use cosmetic
products. Therefore, if anti-melanogenic action of amaranth
turned, not simply providingan squalene’s additional
resource, it may be a potential good source for functional
cosmetics.
To determine the effects of AT on melanogenesis,
murine melanocytes were treated with different concent-
rations of AT for 72 h. Among various concentrations and
samples, we selected AC and AD as representative samples
and further experiments were performed.
Figure 3 indicates that AT inhibits melanin synthesis
when compared with control conditions. Especially, 10
µM of AC and AD significantly reduced melanin content
without cell toxicity in melan-a cells. Additionally, their
anti-melanogenic effect was stronger than that of arbutin,
a widely known whitening agent. These data suggest that
AT affects melanogenesis and regulates melanin synthesis.
Therefore, it is very interesting because it has possibility
for skin whitening.
Tyrosinase is the main enzyme in melanin synthesis. It
triggers the melanin synthesis pathway and affects mela-
nogenic enzymes. It converts L-tyrosine to melanin
polymers and stimulates TRP-1 and TRP-2 expression.34
Therefore, tyrosinase activity was measured in cell or
cell-free conditions.35 To investigate whether AT inhibits
tyrosinase activity, we estimated tyrosinase activity in
vitro. Kojic acid, a well-known tyrosinase inhibitor, was
used as a positive control. Compared with control, AC
decreased the activity of mushroom tyrosinase, but not
significantly. However, the AD treated group showed a
significant decrease in tyrosinase activity. The level of
inhibition of tyrosinase activity by A D was similar to that
of Kojic acid (Fig. 4). Our results indicate that AD
inhibits melanin formation by reducing tyrosinase activity.
Additionally, its effect is similar to that of kojic acid,
which is a well-known compound that decreases melanin
synthesis by reducing tyrosinase activity.36
Interestingly, the effect of AT on the expression of
Fig. 2. HPLC chromatogram for the determination of myo-inositol in Amaranth ushypocondriacus (A.D). A. Chromatogramof myo-inositol B. Chromatogram of AD.
Fig. 3. Effects of Amaranth seed extract (AT) on melanin contentand cell viability in melan-a cells. Melanin contents (A) and cellviability (B) were measured. CTL indicates normal conditionsand Arbu indicate the use of arbutin as a positive control (μM).All sample (μg/mL) were treated for 72 h. Data are expressed asmean ± S.D. of three experiments. * p < 0.05, ** p < 0.01, and*** p < 0.001 compared with control.
Fig. 4. Effects of AT on tyrosinase activity in melan-a cells.Melan-a cell originated tyrosinase activity was measured. CTLindicates normal conditions and KA indicate the use of kojic acidas a positive control (μM). Each samples were treated with μg/mL. The results are expressed as mean ± S.D. of three experi-ments (* p < 0.05).
172 Natural Product Sciences
tyrosinase and/or TRP-2 was significantly stronger than
that of arbutin.
To determine the mechanism by which AT affects
melanin synthesis, we assessed the expression of mela-
nogenic enzymes (tyrosinase, TRP-1, TRP-2, and MITF)
by western blot analysis. We performed a western blot
analysis after treatment with AC and AD for 3 days. As
shown in Fig. 5, the expression of melanogenic enzymes
decreased in a dose dependent manner after AT treatment
(Fig. 5). AC and AD reduced the expression of mela-
nogenic enzymes such as TRP-1, TRP-2, and tyrosinase,
resulting in a decrease in melanogenesis.AT significantly
decreased TRP-2 and tyrosinase levels when compared
with control conditions. Especially, A.C reduced the
expression level of TRP-2 to 35.2%. Although there was
no effect on MITF expression, the expression of other
melanogenic enzymes was decreased by AT treatment.
Thus, we can conclude that AT decreases melanin
synthesis by downregulating the expression of TRP-2 and
tyrosinase. And, continuously, to determine an active
component of AT, we tried to make subfraction of AT.
Among them, hexane-soluble and water-soluble subfraction
from AT showed the decrease of melanin in melan-a cells.
It is well soluble in water, therefore, we can suggest that
anti-melanogenic activity of water soluble fraction from
AT is derived from myo-inositol.
This study was designed to determine the anti-mela-
nogenic effects of AT. Among amaranth species, especially,
Amaranth ushybridus significantly reduced the melanin
level in melan-a cells by downregulating melanogenic
enzymes such as tyrosinase and TRP-2.
Fig. 5. Effects of Amaranth ushybridus (A.C) and Amaranth ushypocondriacus (A.D) on the expression of melanogenic enzymes inmelan-a cells. To confirm the expression of melanogenic enzymes, melan-a cells were treated with 0.1, 1 and 10 μg/mL of A.C and A.D.Arbutin was used as a positive control (μM). (A) Western blot analysis. Densitometric analysis of Tyrosinase (B), TRP-1 (C), TRP-2 (D)and MITF (F). Each band was normalized to that of α-tubulin.
Fig. 6. Effects of each fraction of amaranth seed extract (AT) onmelanin content and cell viability in melan-a cells Melanincontents (A) and cell viability (B) were measured. CTL indicatesnormal conditions and Arbu indicate the use of arbutin as apositive control (μM). All sample (μg/mL) were treated for 72 h.Data are expressed as mean ± S.D. of three experiments. *p < 0.05, ** p < 0.01, and *** p < 0.001 compared with control.
Table 1. Contents (μg/mg) of myo-inositol in Amaranth ushypo-condriacus (A.D).
r2 Content
Myo-inositol 0.9955 49.036 ± 1.032
Vol. 22, No. 3, 2016 173
Our results support the view that amaranth species may
be used as a nutricosmetic food as well as a potential
skin-whitening agent. Specially, the amaranth as a
nutricosmetic agent has merits in respective of safety. In
fact, amaranth is one of the major dietary sources in South
America. However, further epidemiological studies are
necessary to determine the safety and impact of amaranth
on the skin. Therefore, further in vivo and clinical studies
are warranted to determine the main active ingredient in
amaranth, which is involved in its skin-whitening property.
In conclusion, this study suggests that amaranth may be
a potential hypo-pigmentatory agent from natural products.
Furthermore, it can be used for nutricosmetical resources.
Acknowledgments
This work was carried out with the support of
“Cooperative Research Program for Agriculture Science
& Technology Development (No. PJ01118803)” Rural
Development Administration, Republic of Korea.
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Received November 5, 2015
Revised February 23, 2016
Accepted February 29, 2016
e·